Engineering lipid vesicles of enhanced intratumoral transport capabilities: correlating liposome characteristics with penetration into human prostate tumor spheroids.

Liposomes have been widely used delivery systems, particularly relevant to the development of cancer therapeutics. Numerous liposome-based drugs are in the clinic or in clinical trials today against multiple tumor types; however, systematic studies of liposome interactions with solid or metastatic tumor nodules are scarce. This study is describing the in vitro interaction between liposomes and avascular human prostate (LNCaP-LN3) tumor spheroids. The ability of fluorescently labelled liposomal delivery systems of varying physicochemical characteristics to penetrate within multicellular tumor spheroids has been investigated by confocal laser scanning microscopy. A variety of liposome characteristics and experimental parameters were investigated, including lipid bilayer composition, duration of liposome-spheroid interaction, mean liposome size, steric stabilization of liposomes. Electrostatic binding between cationic liposomes and spheroids was very efficient; however, it impeded any significant penetration of the vesicles within deeper layers of the tumor spheroid. Small unilamellar liposomes of neutral surface character did not bind as efficiently but exhibited enhanced penetrative transport capabilities closer to the tumor core. Polymer-coated (sterically stabilised) liposomes exhibited almost no interaction with the spheroid, indicating that their limited diffusion within avascular tissues may be a limiting step for their use against micrometastases. Multicellular tumor spheroids were used as models of solid tumor interstitium relevant to delivery systems able to extravasate from the microcapillaries or as models of prevascularized micrometastases. This study illustrates that interactions between liposomes and other drug delivery systems with multicellular tumor spheroids can offer critically important information with respect to optimizing solid or micrometastatic tumor delivery and targeting strategies.

Liposome-mediated radiotherapeutics within avascular tumor spheroids: comparative dosimetry study for various radionuclides, liposome systems, and a targeting antibody.

UNLABELLED: Absorbed dose profiles within tumor spheroids simulating avascular micrometastases have been calculated for a variety of liposome- and antibody-radionuclide combinations to assess the anticipated therapeutic efficacy based on the intratumoral distribution of the carrier systems within the spheroid model. METHODS: Experiments studying the targeting and diffusion capability of the most clinically relevant liposome systems and the anti-PSMA (prostate-specific membrane antigen) antibody J591 within spheroids of the prostate cancer cell line LNCaP (diameter, 150-200 mum) have been performed. The intratumoral biodistribution data were then used as the input to obtain absorbed dose profiles within the tumor spheroid mass. The dosimetric analysis was performed for a variety of medium- and high-energy beta-emitting radionuclides ((32)P, (90)Y, (188)Re, (67)Cu, (131)I) and 2 low-energy Auger or conversion electron emitters ((123)I, (125)I) following the point-kernel convolution method in the continuous slowing-down approximation. RESULTS: Relative absorbed dose distribution calculations as a function of the distance from the rim of the spheroids are presented. For all liposome systems studied, the SUV-DMPC-chol (small unilamellar vesicle-dimyristoyl-phosphatidylcholine-cholesterol) was most efficient in penetrating deeper within the spheroids. For the beta-emitters it delivered its maximum absorbed dose (D(max)) at 40- to 50-microm depth, exhibiting an almost flat absorbed dose profile beyond that point, as is evident by the high absorbed dose value at the center of the spheroid (D(core)), D(core)/D(max) > 0.9; the respective values for the J591 antibody were 20 mum and 0.85. The Auger or conversion emitters resulted in the most heterogeneous absorbed dose distribution; the ratio D(core)/D(max) fell to 0.4 for the SUV-DMPC-chol and to 0.4-0.5 for the antibody. In general, a 2- to 10-fold "cross-fire"-related increase of the core absorbed dose was observed. For liposomes exhibiting high binding capacity (3beta-[N-(N',N']-dimethylaminoethane)carbamoyl]cholesterol [DC-chol]), however, the low-energy emitters deliver up to a 40% higher D(max) relative to the beta-emitters. The surface characteristics of liposomes appear to have a noticeable influence on the absorbed dose profiles. The use of neutral (DMPC-chol) versus cationic (DC-chol) lipids resulted in up to a 10-fold increase of D(core)/D(max) depending on the radionuclide. Changing the cationic lipid used to N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate also had a notable influence (up to a 6-fold increase), whereas the effect of fusogenic lipids (dioleoylphosphatidylcholine) was found to be much smaller. CONCLUSION: It is possible to engineer liposome systems that are particularly effective in delivering an almost uniform absorbed dose profile at the central region of micrometastatic tumors, provided that conjugates with the appropriate radionuclides are constructed. In view of the passive means of diffusion of liposomes within solid tumors, it is suggested that they may effectively complement an antibody-based therapeutic regime against micrometastatic tumors, leading to cytotoxic absorbed dose levels throughout the entire tumor volume--thus, hindering tumor recurrence.

Binding and interstitial penetration of liposomes within avascular tumor spheroids.

The liposomal delivery of cancer therapeutics, including gene therapy vectors, is an area of intense study. Poor penetration of liposomes into interstitial tumor spaces remains a problem, however. In this work, the penetration of different liposomal formulations into prostate carcinoma spheroids was examined. Spheroid penetration was assessed by confocal microscopy of fluorescently labeled liposomes. The impact of liposomal surface charge, mean diameter, lipid bilayer fluidity and fusogenicity on spheroid penetration was examined. A variety of different liposome systems relevant to clinical or preclinical protocols have been studied, including classical zwitterionic (DMPC:chol) and sterically stabilized liposomes (DMPC:chol:DOPE-PEG2000), both used clinically, and cationic liposomes (DMPC:DOPE:DC-chol and DOTAP), forming the basis of the vast majority of nonviral gene transfer vectors tested in various cancer trials. Surface interactions between strongly cationic vesicles and the tumor cells led to an electrostatically derived binding-site barrier effect, inhibiting further association of the delivery systems with the tumor spheroids (DMPC:DC-chol). However, inclusion of the fusogenic lipid DOPE and use of a cationic lipid of lower surface charge density (DOTAP instead of DC-chol) led to improvements in the observed intratumoral distribution characteristics. Sterically stabilized liposomes did not interact with the tumor spheroids, whereas small unilamellar classical liposomes exhibit extensive distribution deeper into the tumor volume. Engineering liposomal delivery systems with a relatively low charge molar ratio and enhanced fusogenicity, or electrostatically neutral liposomes with fluid bilayers, offered enhanced intratumoral penetration. This study shows that a delicate balance exists between the strong affinity of delivery systems for the tumor cells and the efficient penetration and distribution within the tumor mass, similar to previous work studying targeted delivery by ligand-receptor interactions of monoclonal antibodies. Structure-function relationships from the interaction of different liposome systems with 3-dimensional tumor spheroids can lead to construction of delivery systems able to target efficiently and penetrate deeper within the tumor interstitium and act as a screening tool for a variety of therapeutics against cancer.